ANTIBACTERIAL EFFECT OF AQUEOUS EXTRACT OF ALOE VERA AGAINST THE GRAM POSITIVE BACTERIA STAPHYLOCOCCUS AUREUS IN MEDANI CITY GEZIRA STATE – SUDAN 2018

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Yasir et al. World Journal of Pharmaceutical Research

ANTIBACTERIAL EFFECT OF AQUEOUS EXTRACT OF ALOE

VERA AGAINST THE GRAM-POSITIVE BACTERIA

STAPHYLOCOCCUS AUREUS IN MEDANI CITY - GEZIRA

STATE – SUDAN- 2018

Yasir Hakim1*, Dalia Hamza2, A. KH Khalil3, Abdalla Khalid4

Assistant Professor of Pathology with MD Pathology, Head Unit of Microbiology,

Department of Basic Medical Science, College of Medicine, Dar Al Uloom University,

Riyadh, Kingdom of Saudi Arabia & Sennar University, International College of Medicine,

Sudan.

ABSTRACT

The plant Aloe vera was used historically as a topical to heal wounds,

various skin conditions and orally as a laxative. The Gram-positive

bacterium Staphylococcus aureus is considered to be the most

pathogenic species of the genus Staphylococcus, being implicated in

both community-acquired and nosocomial infections. The present

investigation was undertaken at the University of Gezira, Center of

Plant Pathology, during the year 2018. The aim of the study was to

investigate the effect of Aloe vera on Staphylococcus aureus as

antibacterial activity of aqueous and alcoholic extracts of Aloe vera on

inhibiting the growth of the Staphylococcus aureus against a known

Antibiotics (Gentamycin) as appositive control. Three concentrations

of aqueous extract of Aloe vera and the Gentamycin, (25, 50 and

100%) were tested. The aqueous suspensions of the dried Aloe vera

extracts were screened for their anti-Staphylococcus aureus activity

using the agar-disc diffusion method. The results obtained indicated

that the highest concentration of Aloe vera aqueous extract (100%)

used in this study had the highest inhibitory effects (18.2 mm) against

the tested bacterium, while the other two concentrations (50 % and 25%) showed an

inhibition zones of 15 mm and 12 mm, respectively. For the positive control (Gentamycin)

the highest inhibition zone 16.5 mm was obtained with the higher concentration (100 %). The

World Journal of Pharmaceutical Research

SJIF Impact Factor 8.074

Volume 7, Issue 12, 18-34. Research Article ISSN 2277– 7105

Article Received on 22 April 2018,

Revised on 12 May 2018, Accepted on 03 June 2018,

DOI: 10.20959/wjpr201812-12607

*Corresponding Author

Yasir Hakim

Assistant Professor of

Pathology with MD

Pathology, Head Unit of

Microbiology, Department

of Basic Medical Science,

College of Medicine, Dar

Al Uloom University,

Riyadh, Kingdom of Saudi

Arabia & Sennar

University, International

College of Medicine,

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other concentrations (25 and 50 % showed inhibition zones of 7.75 and 6 mm, respectively.

The study recommended that, further research should be done to clearly identify the active

ingredients of Aloe vera and their other antimicrobial activities.

KEYWORDS:Aloe Vera, Antimicrobial activities, Gentamycin, Staphylococcus aureus.

INTRODUCTION

Aloe vera belongs to the Liliaceae family, of which there are about 360 species. It is a

cactus-like plant that grows readily in hot, dry climates and currently, because of demand, is

cultivated in large quantities. The gel of A. vera was used to treat stomach ailments,

gastrointestinal problems, skin disease, constipation, radiation injury, inflammatory effect,

healing wounds and burns, ulcer and diabetes. A. vera products are mainly for cosmetic,

pharmaceutical, nutraceuticals and food industries. The gel stimulates cell growth and

enhances the restoration of damaged skin. It moisturizes the skin because it has a water

holding capacity. As a drink, it protects the mucous membrane of the stomach especially

when irritated or damaged. A. vera juice is considered helpful for relieving many types of

gastrointestinal irritation and juice products are widely available (Anderson and Phillipson.,

1996).

Staphylococcus aureus poses an important problem in hospitals, nursing homes, and other

health care settings. Serious infections due to these organisms currently necessitate the use of

non -β-lactam antibacterial therapy (Hackbarth and Chambers,1989).Many hospital acquired

MRSA strains are only susceptible to vancomycin (Fitzgerald et al., 2001). Thus, there are

strong concerns about the possible development and spread of vancomycin resistance in

MRSA. Some vancomycin-resistant MRSA strains have been reported since 1996 (EI-Jakee

et al., 2014; ALian et al., 2012). Some necrosis poisons cases occur by strong acids as

H2SO4, it affects skin created necrosis or burns and these allow for bacteria growth. H2SO4 is

one form of strong poison, because the poison's symptoms appear after five minutes from

application on skin. If possible, treatment by Na2Co3 as antidote for H2SO4, but the necrosis

caused by bacterial infection should be treated use drugs. The main constituents of Aloe Vera

gel are mucopolysaccharides (glucomannans, polymannoses, about 10% of total solids),

enzymes, anthranoids, lignin, saponins, vitamins, amino acids (almost 50% of the total

amount consisting of 8 of the 10 essential amino acids) and minerals (quantities not given).

Total solids are in the range of 1.3 to 2%, the rest being water (Vinson et al., 2005). Aloe

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processing of the whole leaf, in which case the product usually also contains appreciable

quantities of the latex material and anthranoids. The anthranoids in whole leaf extracts of

Aloe Vera can however, be reduced to levels below10mg/kg in the product (Reynolds and

Dweck, 1999; Lee et al., 2000; Hu et al., 2003). Oliver (Oliver, 2012) indicates that Aloe

Vera gel is used in veterinary medicine topically to promote wound healing on general skin

wounds in all animals. It has also been recommended as a teat-dip in lactating cows, by intra

mammary administration for (adjuvant) treatment of mastitis or high somatic cell counts, and

by oral route in all food producing species as adjuvant treatment for a number of afflictions

(ranging from anemia to infertility, mastitis and shock (Hu et al., 2003;Oliver,2012).

Medicinal plants according to the World Health Organization (WHO) defines them as herbal

preparations made by introducing plant materials to extraction, fractionation, purification,

concentration, or other physical or biological processes, which may be produced as a basis for

herbal products or for immediate consumption. In human medicine Aloe Vera gel is used

topically to promote wound healing. Oral use as a general tonic for a number of indications,

where scientific proof is outstanding, has also been described. Aloe Vera gel is also widely

used in cosmetics (Ramachandra and Rao, 2008; Subramanian et al., 2006; Saravanan et al.,

2010; Kedarnath et al., 2012). Moreover, Aloe Vera has ulcerogenic activity (Sai et al.,

2014).

OBJECTIVES

 To test the antimicrobial effects of Aloe vera aqueous and alcohol and Antibiotic (control) leaf

extracts on Staphylococcus aureus.

 To determine the effect of Aloe vera leaf extract different concentration on

Staphylococcusaureus.

MATERIALS AND METHODS

Staphylococcus aureus

It was obtained from the microbiological laboratory of the Department of Pathology Medical

lab, Faculty of Medicine, University of Gezira, Wad Medani, Sudan during the period from

January to May, 2018.

Aloe vera plant

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Preparation of Nutrient agar

This was a general-purpose cultured medium for bacteria. It was obtained in a dehydrated

form. The constituent of the medium were beef extract, yeast extract, peptone, sodium

chloride and agar. It was prepared according to the manufactures instruction by suspending

28g in one liter distilled water. The medium was allowed to boil until it was completely

dissolved. The pH of medium was adjusted to pH 7.4±0.2 and then the medium was sterilized

in an autoclave at 121ºC (115b\in²) for 15 min (Harrigan, 1998).

Preparation of the crude extracts

Aloe vera leaf extract to prepare crude extract of fresh Aloe vera whole leaves were washed

with distilled water, chopped into small pieces, air-dried and ground into powder. The Aloe

vera mixed with 80% concentration of ethanol The pulp ethanol mix was then centrifuged at

3000rpm for 10 minutes and the supernatant collected was allowed to evaporate over a dry

oven. The gelatinous extract thus prepared was weighed using distilled water, serial dilutions

of 25g/75ml, 50g/50ml and 100mg (w/v) were made in order to obtain 25%, 50% and 100%

concentrations, respectively.

Preparation of test organism

The nutrient agar were mixed well and poured on the sterile petri plates. The agar media on

petri plates were allowed to set for few minutes. nutrient agar plates were inoculated with

respective bacteria (S.aureus ), and then incubated at 37° C for overnight. Each time, a fresh

bacterial culture was prepared.

Antimicrobial agent

The antibacterial agent gentamicin was dissolved in distilled water. Further dilutions were

made using the same solvent according to CLSI document M100-S18. Gentamicin was used

in the concentrations 25%, 50% and 100%.

Antibacterial activity

Antibacterial activity was measured using paper disc diffusion method, (Method of Saba et

al., 2011) was followed.

The following steps were involved in paper disc diffusion method. The normal agar were

mixed well and poured on the sterile petri plates. The agar media on petri plates were allowed

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The small autoclaved discs of Whatmann filter paper were used. The test organism was

spread on the petri plates by using sterilized glass spreader. During paper-disc diffusion

method, the sterile discs were dipped in the different crude extracts of medicinal plants and

antibiotic drugs with the help of sterilized forceps and placed on the Petri plates. Distilled

water was used as a control to check the comparison of antibacterial activity with different

crude extracts of medicinal plants. The petri plates were sealed with para film.

Then, the petri plates were left at room temperature for 30 minute, to allow the diffusion of

the test sample and then incubated at 37° C for overnight. The diameter of the zones of

inhibition were measured in cm.

Statistical analysis

The obtained data was statistically analyzed by computer software MSTATC according to

analysis of variance (ANOVA); Duncan's Multiple Range Test was used for mean separation.

RESULTS

1. Two days post inoculation

The results depicted in Table (1) and figure (1) indicate that the high concentrations of Aloe

vera aqueous extract (100%) used in this study had the highest inhibitory effects (18.2 mm)

against the tested Staphylococcus aureus. However, this extract showed inhibition action of

12 mm even at minimal concentration (25%) used. The other concentrations of the aqueous

phase (50 %) gave an inhibition zones of 15 mm.

Control (Gentomycin) extract of Aloe vera at all concentrations shows an inhibitory effect

against the Staphylococcus aureus. The highest inhibition zone obtained was 16.5 mm with

the concentration of 100 % Concentrations of 25 and 50 % showed an inhibition zones of

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Table 1: Effect of different Concentration of aqueous and alcoholic extracts of Aloe vera and Antibiotic on inhibition (mm) of Staphylococcus aureus using disc method at two days post inoculation.

Treatments Concentration % Inhibition zones(mm) Mean R1 R2 R3

Ethanol

25 7.5 6 5.8 5.9

50 10 9 9.8 9.4

100 16 14 15 14.5

Aqueous

25 10 13 12 12

50 15.8 16 14 15

100 19.2 18.4 18 18.2

Gentamycin

25 6.5 8.5 7 7.75

50 9 7 5 6

[image:6.595.134.464.134.470.2]

100 17 15 18 16.5

Figure 1: Effect of different Concentration of aqueous and alcoholic extracts of Aloe vera and Antibiotic on inhibition (mm) of Staphylococcus aureus using disc method at two days post inoculation.

2. Three days post inoculation

The results depicted in Table( 2 )and Figure ( 2) indicate that the high concentrations of Aloe

vera aques extract (100%) used in this study had the highest inhibitory effects (16.65 mm)

against the tested Staphylococcus aureus. However, this extract showed inhibition action of

10.5 mm even at minimal concentration (25%) used in this study. The other concentrations of

the aqueous phase (50 %) gave an inhibition zones of 13 mm.

Control (gentamycin) extract of Aloe vera at all concentrations shows an inhibitory effect

against the Staphylococcus aureus. The highest inhibition zone obtained was 16 mm with the

concentration of 100 % Concentrations of 25 and 50 % showed an inhibition zones of 5.95

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Table 2: Effect of different Concentration of aqueous and alcoholic extracts of Aloe vera and Antibiotic on inhibition (mm) of Staphylococcus aureus using disc method at three days post inoculation.

Treatments Concentration % Inhibition zones(mm) Mean R1 R2 R3

Aqueous

25 10 11 10 10.5

50 14 14 12 13

100 18 16.4 16.9 16.65

Gentamycin

25 5 7.3 6.6 5.95

50 8 6 5 5

100 16 15 17 16

Figure 2: Effect of different Concentration of aqueous and alcoholic extracts of Aloe vera and Antibiotic on inhibition (mm) of Staphylococcus aureus using disc method at three days inculation.

3. Four days post inoculation

The results depicted in (Table 3 and Figure 3) indicate that the high concentrations of Aloe

vera aqueous extract (100%) used in this study had the highest inhibitory effects (16 mm)

against the tested microorganisms. However, this extract showed inhibition action of 9 mm

even at minimal concentration (25%) used in this study. The other concentrations of the

aqueous phase (50 %) gave an inhibition zones of 11.5 mm.

Control (Gentomycin) extract of Aloe vera at all concentrations shows an inhibitory effect

against the Staphylococcus aureus. The highest inhibition zone obtained was 14 mm with the

concentration of 100 % Concentrations of 25 and 50 % showed an inhibition zones of 4.5 and

[image:7.595.131.465.133.436.2]

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Table 3: Effect of different Concentration of aqueous and alcoholic extracts of Aloe vera

and Antibiotic on inhibition (mm) of Staphylococcus aureus using disc method at four days post inoculation.

Treatments Concentration% Inhibition zones(mm) Mean R1 R2 R3

Aqueous

25 9 10 8 9

50 12.8 12 11 11.5

100 17 16 16 16

Gentamycin

25 3.5 5 4 4.5

50 7 5 4 4.5

100 15 13 15 14

Figure 3: Effect of different Concentration of aqueous and alcoholic extracts of Aloe vera and Antibiotic on inhibition (mm) of Staphylococcus aureus using disc method at four days post inoculation.

[image:8.595.133.464.134.453.2]

Table 4: Antimicrobial activity of different Concentration preparations of Aloe vera gel aqueous and alcoholic extract on inhibition (mm) of Staphylococcus aureus with antibiotics as reference standard.

Treatments Concentration% Inhibition zones(mm)

Aqueous

25 +

50 ++

100 ++

Gentamycin

25 +

50 ++

100 ++

Antimicrobial activity: No inhibition +, Zone of inhibition ≤8mm in diameter; ++, Zone of

[image:8.595.156.444.599.700.2]

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DISCUSSION

This study showed that aqueous exstract phase of Aloe vera gave better results compared to

the antibiotic phase of the same extract at this study at all concentration tested. The broad

antimicrobial action of the aqueous extract of the Aloe vera used in the study could be

ascribed to the water soluble components which are naturally occurring in the plant materials.

Other workers have reported There was no antimicrobial activity was reported using aqueous

extract of A. vera leaves (Martineze et al.,1996).

The antimicrobial activity of the extracts and their potency was quantitatively assessed by the

presence or absence of inhibition zone and zone diameter. Only alcoholic extract was found

to be a better solvent for extraction of antimicrobially active substances compared to water

and hexane (Ahmad et al., 1998).

(Subramanian et al., 2006) In other studies, the most effective antibiotic for gram positive is

vancomycin than Gentamycin (Hoeger., 2004).

Aloe vera is a potent antimicrobial agent compared with the conventional antibiotics. The

results of the study by Coopoosamy and Magwa., (2007) also revealed that lowest

concentrations of ethyl acetate and ethanol crude extracts of Aloe excels resulted in complete

inhibition of visible growth of pathogenic bacteria compared with the control antibiotics,

chloramphenicol and streptomycin sulfate.

The results of this study disagree with earlier studies that showed that the antibacterial

activity of some Iranian medicinal plants were more significant in the solvent extracts

compared with aqueous extracts in all the plants, indicating that the active principle(s)

responsible for antibacterial activity were more soluble in organic solvents (Babu et al.,

2007). Similarly, the solvent extract of cloves flower was also found to exhibit a significantly

higher inhibitory effect on the caries-inducing properties of Streptococcus mutans compared

with the crude aqueous extracts (Abd Rahim and Gulam.,2006). In other studies the

methanolic and petroleum spirit extracts of Pelargonium essential oils were more potent

antibacterial agents than the steam distilled volatile samples (Lis- Balchin et al.,1998).

Finally, it was observed that the highest concentration of the aqueous and ethanolic extract of

the plant has significant effect on the bactria isolates. They had liger zone of inhibition

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et al. (2006).

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